Study of Metabolic Absorption Rates of DHA-Taurine as a Source of Omega-3 Fatty Acids

SMART, version 2.0 Protocol summary March 2025 Project ID: H-25014363

Study of Metabolic Absorption Rates of DHA-Taurine as a source of omega-3 fatty acids (SMART)

• Background and purpose:
• Omega-3 fatty acids (FA) have been recognised for their health benefits and their functions in preventing various diseases. Some of these, including α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are necessary for biological processes in the human body and are components in the structure of the cell membranes. Previous studies have shown that taking DHA and/or EPA with doses of 1-4 g/day can lower fat levels (triglycerides and cholesterol) in the blood and liver, reduce inflammation and decrease cardiovascular disease (CVD) risk with minimal side effects (fishy aftertaste and burping).
• Omega-3 fatty acids have a structure that cannot be produced in the human body, so these must be consumed in the diet or as supplements. The body then uses these fatty acids to produce anti-inflammatory agents. Therefore, omega-3 fatty acid supplements could be an addition to treatment for many inflammatory diseases, and are being investigated for benefits in treating diseases like diabetes, autoimmune disease, cancer, depression, rheumatoid arthritis, etc. Because of the effects in lowering blood triglycerides, the fatty acids are often recommended for treatment of many cardiometabolic conditions. However, omega-3 fatty acids are usually consumed in forms (e.g. ethyl esters) that require a fully functional mechanism of digestion and absorption of lipids (fat), which is complex, and can be compromised in many conditions, including pancreatitis, cystic fibrosis, small bowel resection, and cancer. A very low fat-diet to support weight loss or CVD management also limits the absorption of omega-3 FA. Therefore, even though these people may greatly benefit from a higher intake of omega-3 FA, the FA are not absorbed through the digestive tract and must either utilize intravenous delivery or additionally use external digestive enzymes, which come with significant costs and risks. One solution is to use omega-3 FA-containing molecules that are absorbed through a different mechanism but can be modified to supply the needed omega-3 FA.
• A recent study in mice, conducted by Grevengoed and colleagues in 2021, studied a naturally occurring metabolite derived from the omega-3 fatty acid docosahexaenoic acid (DHA), and the authors assessed that this metabolite may be responsible for some of the beneficial effects of omega-3 fatty acids. The metabolite is of the N- acyl type taurine (NAT), and consists of the fatty acid DHA bound to a taurine (DHA-T). NATs are fats that are active in biological processes and are found naturally in mammals in tissue, blood and bile. Mouse studies have previously shown that some types of NAT have a positive effect on metabolism, by increasing insulin sensitivity and glucose tolerance, as well as increasing the secretion of glucagon-like peptide 1 (GLP-1). GLP-1 is a hormone secreted from the intestine that, among other things, increases the feeling of satiety and regulates other aspects of metabolism.
• DHA-T is an interesting metabolite in the context of cardiometabolic risk reduction. Grevengoed and colleagues obtained comparable results by feeding mice fish oil as by giving DHA-T directly, suggesting that DHA-T is responsible for the triglyceride-lowering properties of omega-3 fatty acids. In addition, DHA-T shows potential to reduce both the physical risk factors for CVD (by reducing triglyceride levels in the blood) and the behavioral risk factors (by increasing the secretion of GLP-1, which increases satiety and can thus induce weight loss).

The first human trial with a NAT was conducted in 2022, and it was found that oral DHA-containing NAT (DHA-T) was well absorbed with a high fat meal. Furthermore, another experiment in mice showed that NATs were absorbed without dietary fat, and in a model of impaired lipid (fat) absorption, absorption of DHA-T was identical to mice with normal absorption of lipids.

However, the molecular mechanisms of transport, delivery and use of the NAT-derived FA are not yet fully known. It is important to know the underlying molecular mechanisms because it increases the understanding of the condition you want to treat, makes it easier to target the treatment, and avoid side effects.

The ambition of this trial is to determine if DHA-T is better absorbed than a standard DHA supplement form, ethyl esters, without fat in the meal which would potentiate a route of absorption that is impaired in many diseases. This will help us understand mehanisms of absorption for this molecule and the availability of the DHA originated from NAT, which could source as a FA to be used in other biological processes.

• Purpose
• Investigate the following effects of 2 mg/kg DHA-T versus 1,6 mg/kg DHA-EE (which are the same in DHA content) without a meal:
• Plasma DHA level
• Comparison of plasma DHA level delivered via DHA-T versus DHA-EE
• Plasma level of DHA-T
• Experimental design:
• The project is carried out at the Department of Biomedical Sciences, University of Copenhagen, where there is access to the necessary equipment for carrying out the various trial elements. The project is divided into a preliminary study (screening) and two trial days, where the trial participants must undergo an absorption test.
• Trial participants:
• The trial will include a total of 7 healthy male volunteers, between 18 and 30 years of age. The trial participants must have a BMI between BMI 18.5 - 25. The participants must not have taken fish oil supplements in the past 3 months, or eat more than 350 g of fish per week. The participants must not be allergic to any ingredients in the standardized meal, must not smoke or use other nicotine products, and must have a moderate level of activity. The participants must not suffer from liver, biliary or kidney diseases, as well as diabetes or have first-degree family members with diabetes or familial hyperlipidemia / hypercholesterolemia. In addition, the participants must not be taking medication or suffer from other conditions that can be assessed as affecting participation in the trial.
• Experimental setup:
• The trial design is a double-blind, randomized, crossover study comprising 2 separate treatment days with one of the following double-blind interventions:

• Absorption test without a meal with DHA-T
• Absorption test without a meal with DHA-EE
• A study assistant randomizes and mixes the test lipid, so that the project manager is also blinded during the absorption test.
• Screening:
• The preliminary study lasts approx. one hour. The trial participant will have their blood pressure, weight, height, body fat percentage measured, and have blood samples and a urine sample taken. Any medication use and medical history will be noted. If the trial participant can participate in the project, the trial days will be planned.
• The trial days:
• The two test days last approximately 9 hours each, of which the absorption test lasts 480 minutes. Participants must fast from 10:00 PM the evening before the test days and are provided with dinner and an evening snack to be consumed the evening before the test day. 72 hours before, the participants must refrain from strenuous exercise, eating more than normal, alcohol, and 1 week before the test day, the participants must refrain from eating fish.

On the day of the test, a peripheral venous catheter is inserted in the elbow bend, from which blood samples are taken. Throughout the test, the test participant will have the volume of the gallbladder measured regularly using ultrasound, and have blood samples taken. After the first ultrasound measurement and taking fasting blood samples, the test subject consumes two 10 ml solutions with a weight-adjusted DHA-T or DHA-EE in one of them, as well as 500 ml of water with lemon. After the 180 minutes have passed, a light, low-fat meal is served to the test subjects, and after 360 minutes, a snack is offered.

• Research biobank:
• The blood samples will be analyzed at Rigshospitalet Hospital and at the University of Copenhagen. The biological material will be stored in a research biobank until the project's expected end in October 2025, in case there is a need to repeat faulty analyses or if additional analyses are needed. Excess biological material will be stored for future research after 2025. Throughout this period, data protection regulations will continue to be complied with; however, the Scientific Ethics Committee may waive the requirement to obtain consent from the trial participants for new analyses.
• Side effects, risks and disadvantages of the trial:

In connection with each test day, the participants will loose a maximum of 275 ml of blood. This is about a half of what is taken in a single blood donation. Iron treatment is offered after participation in order to rebuild the blood percentage after the last day of the experiment. The 10-hour fast up to the experiment is part of the natural nocturnal fast (10 p.m. - 8 a.m.), and is thus neither associated with discomfort nor danger. There is a chance to experience brief discomfort or pain as well as bruising when inserting the catheter in the elbow bend. There is a small risk of superficial infection when inserting the IV (and any other penetration of the skin and blood vessels with sharp/pointed objects). Any infection can be treated with antibiotics, or with anti-inflammatory cream if it is a superficial local irritation. The risk of superficial infection is small and is minimized by following clinical standards for inserting the IV and other sterile procedures, including double wiping of the involved skin area with disinfectant alcohol. Even though very unlikely, with every ingestion comes a certain risk for an allergic reaction. Therefore, an emergency anaphylaxis kit will be available in the room during the study. DHA-T is a naturally occurring metabolite that is formed in all humans when omega-3 fatty acids are broken down. It is not a drug. The dose of DHA-T given in this trial corresponds approximately to the amount of omega-3 fatty acids one normally consumes from, for example, fish oil dietary supplements, which is not associated with any risk. The body itself converts the omega-3 fatty acid to DHA-T, and the investigators therefore do not expect there to be any side effects from consuming the metabolite. However, there may be unforeseen side effects or effects, as with any scientific trial. DHA-T will only be handled by authorized personnel before it is given to the test subjects.

The dose of DHA-EE is matched to the number of DHA molecules in the DHA-T dose, and DHA-EE is a common dietary supplement form of DHA. The standardized meal will be very low on fat, which should not cause any unease.

• Access to trial results:
• If participants wish to receive information about the overall results of the studies, this can be stated on the consent form or by later contacting the investigators.
• Dissemination:
• The project results or parts of the project will be prepared into one or more articles for publication in one or more international scientific journals. Negative as well as inconclusive and positive experimental results will be published.
• Economy:
• The initiative for the trial comes solely from employees at the Department of Biomedical Sciences, University of Copenhagen, who have no financial interest in the conduct or results of the trial. The principal investigator and the responsible physician is Lærke Bruun Madsen MD. Ana Pezo, MD, research assistant, is responsible for the everyday management of the project.

The trial is funded by a research grant from the Novo Nordisk Foundation (NNF), awarded to Trisha Grevengoed (NNF21OC0067271). The grant from NNF is paid to the Department of Biomedical Sciences, University of Copenhagen, and is used for operating expenses, utensils, screening blood samples, bioanalytical assistance and compensation for trial participants. The project is initiated by Associate Professor Trisha J. Grevengoed, PhD and trial leader Lærke Bruun Madsen, MD, Department of Biomedical Sciences, University of Copenhagen. Ana Pezo, MD, is the project manager. Neither the research group behind the project nor NNF have any financial interests in the execution or results of the project.

• Scientific ethics statement:
• Before any protocol-related procedures, the potential study participant is offered an oral information interview. At least one week before this visit, the participant will receive written information about the project along with the brochure "Forsøgspersoners rettigheder i et sundhedsvidenskabeligt forskningsprojekt" and is informed about the possibility of bringing a witness. At the oral information interview an investigator from the project group will explain the purpose, procedure, and possible side effects of the study in undisturbed and confidential surroundings. The potential trial participant is offered 24 hours to reflect, and if the person concerned is still interested in participating in the trial, the written consent declaration will be signed by the trial participant and the trial personnel. The protocol complies with the Helsinki Declaration II. Participants are covered by the Act on Complaints and Compensation within the Health Service and can report damages to the Patient Insurance. Personally identifiable data and samples are kept confidential. All participants are assigned a trial number and will only appear on data sheets and blood sample tubes with initials and trial number. As biological material is collected (in the form of blood samples), a research biobank is established and notified to the Danish Data Protection Agency. The samples will be stored for up to 15 years after the end of the trial for the purpose of repeating error analyses and measuring parameters other than those currently planned. The latter will require renewed ethical approval (the Danish Ethics Committee may, however, grant an exemption from this requirement). The samples will be destroyed after the end of the biobank. Trial participants can have their material destroyed at any time if desired. No information from trial participants' patient records will be disclosed to trial administrators unless permission is specifically requested.

The trial may be terminated at any time if the trial participant wishes to withdraw, or if extraordinary circumstances make it impossible to complete the trial. In the event of termination of the trial by us, the participant will be informed of the reason for the termination, but the trial participant does not have to justify any termination.

• Recruitment and reimbursement of expenses for trial subjects:
• Paticipants are recruited throught advertisements regarding the project, which will be published via free newspapers such as MetroXpress, as well as via www.forsøgsperson.dk, other relevant websites and/or by distribution of flyers about the project. Written information about the study is distributed to all potential participants who express interest in study participation.

A disadvantage compensation of DKK 1,500 is paid to trial participants at the end of the trial (taxed as B-income). The disadvantage compensation is paid from the project fund account to the participant's NEM account when the study process has been completed for the individual participant. If a trial participant chooses to withdraw from the trial before it is completed, the amount of the disadvantage compensation will correspond to the current attendance time (DKK 500 per completed trial day).

Inclusion criteria

• Male
• Healthy
• Age between 18 and 30 years
• Body mass index between 18.5-25 kg/m2
• Informed consent
• Moderate level of physical activity

Exclusion criteria

• Use of fish-oil/omega-3 FA supplements within the last 3 months
• Regular tobacco smoking or use of other nicotine-containing products
• Allergy or intolerance to ingredients included in the standardised meals
• Weekly intake of fish >350 g (23)
• First-degree relatives with diabetes and/or glycated haemoglobin (HbA1c) >48 mmol/mol, familial hypercholesterolemia/hyperlipidaemia
• Anaemia (haemoglobin below 8.3 mmol/L)
• Alanine aminotransferase (ALAT) and/or aspartate aminotransferase (ASAT) >2 times upper normal values (Normal values: ALAT < 70 U/L, ASAT <45 U/L)
• Nephropathy (serum creatinine >105 μmol/L) and/or albuminuria (>30 mg/g albumin in urine))
• History of hepatobiliary or gastrointestinal disorder(s)
• Any physical or psychological condition, or ongoing medication, that the investigator evaluates would interfere with trial participation, including any acute or chronic illnesses